*NCBI Gene # or RefSeq#: *Protein ID (NP or XP #) or Wolbachia#: 243277 *Organism (including strain): Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961) Etiologic Risk Group (see link below): Risk Group lv 2 (moderate individual risk, limited community risk) */Disease Information (sort of like the Intro to your Mini Research Write up): Cholera is an acute, diarrheal illness caused by infection of the intestine with the bacterium Vibrio cholerae. Globally, cholera cases have increased steadily since 2005 and the disease still occurs in many places including Africa, Southeast Asia, and Haiti.An estimated 3-5 million cases and over 100,000 deaths occur each year around the world. The infection is often mild or without symptoms, but can sometimes be severe. Approximately one in 10 (5-10%) infected persons will have severe disease characterized by profuse watery diarrhea, vomiting, and leg cramps. In these people, rapid loss of body fluids leads to dehydration and shock. Without treatment, death can occur within hours. Link to TDR Targets page (if present): Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): http://www.ncbi.nlm.nih.gov/gene/?term=CAI-1+autoinducer+synthase%2FcqsA ` Essentiality of this protein: Quorum sensing one another and to regulate a wide variety of physiological activities
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer Complex of proteins?: Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB ---- Query Coverage: ---- Max % Identities: ---- % Positives ---- Chain used for homology:
Current Inhibitors: neoglycoprotein Expression Information (has it been expressed in bacterial cells): Escherichia coli Purification Method: Escherichia coli overexpressing V. harveyi cqsA (WN1327) was grown overnight in LB with kanamycin at 30°C with shaking. The culture was diluted 1000-fold in M9 minimal salts medium (Sigma) supplemented with 0.05% (w/v) leucine, 2 mM MgCl2, 0.1 mM CaCl2, 0.01% (w/v) thiamine and 0.4% (w/v) glucose with 10 mg l−1 kanamycin. The culture was incubated overnight at 30°C with shaking. Cells were removed by centrifugation and the cleared fluid was extracted by DCM. The extract was carefully concentrated without heating to a small volume (5 ml) in a Rotovap. The concentrated extract was fractionated using HPLC (see Supporting information). Each HPLC fraction was assayed in triplicate for V. harveyi CqsS agonist activity using the reporter strain JMH626 as described above. Fractions containing activity were further characterized. Image of protein (PyMol with features delineated and shown separately):
SKNKNIIRLS LNSDVNDEQI AKIIEVCSDA VNYGDFYFR *length of your protein in Amino Acids: 389 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 43593.6 Molar Extinction coefficient of your protein at 280 nm wavelength: 36370 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only): *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene # or RefSeq#:
*Protein ID (NP or XP #) or Wolbachia#: 243277
*Organism (including strain): Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Etiologic Risk Group (see link below): Risk Group lv 2 (moderate individual risk, limited community risk)
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Cholera is an acute, diarrheal illness caused by infection of the intestine with the bacterium Vibrio cholerae. Globally, cholera cases have increased steadily since 2005 and the disease still occurs in many places including Africa, Southeast Asia, and Haiti.An estimated 3-5 million cases and over 100,000 deaths occur each year around the world. The infection is often mild or without symptoms, but can sometimes be severe. Approximately one in 10 (5-10%) infected persons will have severe disease characterized by profuse watery diarrhea, vomiting, and leg cramps. In these people, rapid loss of body fluids leads to dehydration and shock. Without treatment, death can occur within hours.
Link to TDR Targets page (if present):
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):
http://www.ncbi.nlm.nih.gov/gene/?term=CAI-1+autoinducer+synthase%2FcqsA `
Essentiality of this protein: Quorum sensing one another and to regulate a wide variety of physiological activities
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077805/
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer
Complex of proteins?:
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
*EC#: 2.3.1.194
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=2.3.1.194
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
**http://www.sigmaaldrich.com/catalog/search?term=CAI-1+autoinducer+synthase%2FcqsA&interface=All&N=0+220003052&mode=partialmax&lang=en®ion=US&focus=site**
-- -or link (or citation) to paper that contains assay information-- links to assay reagents (substrates) pages.
**http://www.sciencedirect.com/science/article/pii/S002228360900881X**
--- List cost and quantity of substrate reagents, supplier, and catalog #Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors: neoglycoprotein
Expression Information (has it been expressed in bacterial cells): Escherichia coli
Purification Method:
Escherichia coli overexpressing V. harveyi cqsA (WN1327) was grown overnight in LB with kanamycin at 30°C with shaking. The culture was diluted 1000-fold in M9 minimal salts medium (Sigma) supplemented with 0.05% (w/v) leucine, 2 mM MgCl2, 0.1 mM CaCl2, 0.01% (w/v) thiamine and 0.4% (w/v) glucose with 10 mg l−1 kanamycin. The culture was incubated overnight at 30°C with shaking. Cells were removed by centrifugation and the cleared fluid was extracted by DCM. The extract was carefully concentrated without heating to a small volume (5 ml) in a Rotovap. The concentrated extract was fractionated using HPLC (see Supporting information). Each HPLC fraction was assayed in triplicate for V. harveyi CqsS agonist activity using the reporter strain JMH626 as described above. Fractions containing activity were further characterized.
Image of protein (PyMol with features delineated and shown separately):
http://www.rcsb.org/pdb/explore/explore.do?structureId=2WK7
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MNKPQLPDFI QNKIDHYIEN YFDINKNGKH LVLGKQASPD DIILQSNDYL
ALANHPLIKA RLAKSLLEEQ QSLFMSASFL QNDYDKPMIE KRLAKFTGFD
ECLLSQSGWN ANVGLLQTIC QPNTNVYIDF FAHMSLWEGA RYANAQAHPF
MHNNCDHLRM LIQRHGPGII VVDSIYSTLG TIAPLAELVN ISKEFGCALL
VDESHSLGTH GPNGAGLLAE LGLTREVHFM TASLAKTFAY RAGAIWCNNE
VNRCVPFISY PAIFSSTLLP YEAAGLETTL EIIESADNRR QHLDRMARKL
RIGLSQLGLT IRSESQIIGL ETGDERNTEK VRDYLESNGV FGSVFCRPAT
SKNKNIIRLS LNSDVNDEQI AKIIEVCSDA VNYGDFYFR
*length of your protein in Amino Acids:
389
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
43593.6
Molar Extinction coefficient of your protein at 280 nm wavelength:
36370
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**